Objective: To isolate and screen Actinomycetes from Lagos Lagoon soil sediments for production of bioactive metabolites. Methods: Sediment samples were collected from four different locations of Lagos Lagoon and were dried for 2 weeks after which the Actinomycetes were isolated by serial dilution using the spread plate method on starch casein and Kuster's agar supplemented with 80 μg/ mL cycloheximide to prevent fungal growth. The plates were incubated at 28 °C for 1-2 weeks. Isolates were selected based on their colonial characteristics as well as their Gram's reaction and subcultured using the same media for isolation until pure cultures were obtained and incubated at 28 °C for 3 d. Thereafter, they were inoculated into starch casein and Kuster's broth media and incubated for 8 d. The secondary metabolites were screened for antimicrobial activity against the following microorganisms: methicillin resistant Staphylococcus aureus, Staphylococcus aureus ATCC 29213, Escherichia coli ATCC 29522, Pseudomonas aeruginosa ATCC 27853, Candida albicans and Enterococcus faecalis ATCC 29212. Coagulasenegative staphylococci isolated from HIV patients were also used (Staphylococcus warneri, Staphylococcus xylosus and Staphylococcus epidermidis). The antimicrobial metabolites of the Actinomycetes isolates were identified using gas chromatography (GC). Results: Crude extracts of isolates showed antimicrobial activity against some of the test organisms. The GC data analysis showed the antibiotic profile of these isolates. Conclusions: Analysis of the crude extracts of the isolates using GC method, revealed the presence of antibiotics including an anticholinergic hyoscyamine among other conclusions.